During the past 15 years, we have carried out several different systematic screens for small regulatory RNA genes in Escherichia coli. These screens have included computational searches for conservation of intergenic regions and direct detection after size selection or co-immunoprecipitation with the RNA binding protein Hfq. We recently examined small RNA expression using deep sequencing to further extend our identification of small RNAs, particularly antisense RNAs (1). A major focus for the group has been to elucidate the functions of the small RNAs we and others have identified. Early on we showed that the OxyS RNA, whose expression is induced in response to oxidative stress, acts to repress translation through limited base pairing with target mRNAs. We discovered OxyS action is dependent on the Sm-like Hfq protein, which acts as a chaperone to facilitate OxyS RNA base pairing with its target mRNAs (2). Recently we carried out extensive mutational studies of Hfq (3). This analysis revealed that amino acids on three different RNA interaction surfaces--the proximal face, the distal face and the rim of the doughnut-shaped protein--differentially impact Hfq association with small RNAs and their mRNA targets (4). It is now clear that Hfq-binding small RNAs, which act through limited base pairing, are integral to many different stress responses in E. coli and other bacteria (5). For example, we showed that the Spot 42 RNA, whose levels are highest when glucose is present, plays a broad role in catabolite repression by directly repressing genes involved in central and secondary metabolism, redox balancing, and the consumption of diverse nonpreferred carbon sources. Similarly, we discovered that a Sigma(E)-dependent small RNA, MicL, transcribed from a promoter located within the coding sequence of the cutC gene represses synthesis of the lipoprotein Lpp, the most abundant protein in the cell, to oppose membrane stress. We found that the copper sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs (6). Most recently, we have characterized a set of small RNAs expressed from a locus we have denoted sdsN. Two longer sRNAs, SdsN137 and SdsN178 are transcribed from two Sigma(S)-dependent promoters but share the same terminator. Whole genome expression analysis after pulse overexpression of SdsN137 and assays of lacZ fusions revealed that the SdsN137 directly represses the synthesis of the nitroreductase NfsA, which catalyzes the reduction of the nitrogroup (NO2) in nitroaromatic compounds and the flavohemoglobin HmpA, which has aerobic nitric oxide (NO) dioxygenase activity. Consistent with this regulation, SdsN137 confers resistance to nitrofurans. Interestingly, SdsN178 is defective at regulating the above targets due to unusual binding to the Hfq protein, but cleavage leads to a shorter form, SdsN124, able to repress nfsA and hmpA. In addition to small RNAs that act via limited base pairing, we have been interested regulatory RNAs that act by other mechanisms. For example, early work showed that the 6S RNA binds to and modulates RNA polymerase by mimicking the structure of an open promoter. In a more recent study, we discovered that a broadly conserved RNA structure motif, the yybP-ykoY motif, found in the 5-UTR of the mntP gene encoding a manganese exporter directly binds manganese, resulting in a conformation that liberates the ribosome-binding site (8). Remarkably, we were able to recapitulate the effect of manganese-dependent activation of translation in vitro. We also found that the yybP-ykoY motif responds directly to manganese ions in Bacillus subtilis. The identification of the yybP-ykoY motif as a manganese ion sensor suggests the genes that are preceded by this motif, and encode a diverse set of poorly characterized membrane proteins, have roles in metal homeostasis. Studies to further characterize other Hfq-binding RNAs and their evolution as well as antisense RNAs and small RNAs that act in ways other than base pairing are ongoing.